GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil; Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Sao Paulo, Brazil.
J Proteomics. 2021 Sep 30;248:104355. doi: 10.1016/j.jprot.2021.104355. Epub 2021 Aug 24.
A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of formerly N-glycosylated peptides (FNGPs). LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases. SIGNIFICANCE: We describe a novel method termed limited deglycosylation assay (LDA), to probe conformational changes of glycoproteins on a systems-wide scale. This method improves the current toolbox of structural proteomics by combining site and conformational-specific PNGase F enzymatic activity with large scale quantitative proteomics. X-ray crystallography, nuclear magnetic resonance spectroscopy and cryoEM techniques are the major techniques applied to elucidate macromolecule structures. However, the size and heterogeneity of the oligosaccharide chains poses several challenges to the applications of these techniques to glycoproteins. The LDA method presented here, can be applied to a range of pathophysiological conditions and expanded to investigate PTMs-mediated structural changes in complex proteomes.
一种新的方法来探测糖蛋白构象变化在系统范围内，称为有限糖基化分析（LDA），被描述。该方法通过常用的肽：N-糖苷酶 F（PNGase F）酶测量天然折叠蛋白上 N-糖基化的差异去糖基化率，这反过来又反映了它们在蛋白质表面的空间呈现和溶剂暴露，最终反映了糖蛋白的构象。LDA 包括 1）在天然条件下进行蛋白质水平的 N-去糖基化，2）胰蛋白酶消化，3）糖肽富集，4）肽水平的 N-去糖基化和 5）以前的 N-糖基化肽（FNGP）的定量 MS 分析。LDA 最初是在牛核糖核酸酶 B 和胎球蛋白的基础上开发的，并优化了实验条件。然后，该方法应用于 LLC-MK2 上皮细胞糖蛋白提取物，用二硫苏糖醇处理诱导内质网应激，促进蛋白质错误折叠。来自 LDA 和 3D 结构分析的数据表明，糖蛋白在 ER 应激下主要在环/转折处发生结构变化，以 Ephrin-A5、GALNT10、PVR 和 BCAM 的详细分析为例。这些结果表明，LDA 准确地报告了在受控处理方案下诱导的糖蛋白系统范围构象变化。因此，LDA 为研究与急性和慢性疾病相关的一系列其他生理和病理生理条件下的糖蛋白结构变化开辟了途径。意义：我们描述了一种新的方法，称为有限糖基化分析（LDA），用于探测糖蛋白在系统范围内的构象变化。该方法通过将位点和构象特异性 PNGase F 酶活性与大规模定量蛋白质组学相结合，改进了现有的结构蛋白质组学工具包。X 射线晶体学、核磁共振波谱学和 cryoEM 技术是阐明大分子结构的主要技术。然而，寡糖链的大小和异质性对这些技术在糖蛋白中的应用提出了一些挑战。这里提出的 LDA 方法可应用于一系列病理生理条件，并扩展到研究复杂蛋白质组中 PTM 介导的结构变化。
